Clock genes:Their role in colorectal cancer

Clock genes create a complicated molecular time-keeping system consisting of multiple positive and negative feedback loops at transcriptional and translational levels. This circadian system coordinates and regulates multiple cellular procedures implicated in cancer development such as metabolism, cell cycle and DNA damage response. Recent data support that molecules such as CLOCK1, BMAL1 and PER and CRY proteins have various effects on c-Myc/p21 and Wnt/β-catenin pathways and influence multiple steps of DNA damage response playing a critical role in the preservation of genomic integrity in normal and cancer cells. Notably, all these events have already been related to the development and progression of colorectal cancer (CRC). Recent data highlight critical correlations between clock genes’ expression and pathogenesis, progression, aggressiveness and prognosis of CRC. Increased expression of positive regulators of this circadian system such as BMAL1 has been related to decrease overall survival while decreased expression of negative regulators such as PER2 and PER3 is connected with poorer differentiation, increased aggressiveness and worse prognosis. The implications of these molecules in DNA repair systems explain their involvement in the development of CRC but at the same time provide us with novel targets for modern therapeutic approaches for patients with advanced CRC.

Effects of Chronotherapy of Benazepril on the Diurnal Profile of RAAS and Clock Genes in the Kidney of 5/6 Nephrectomy Rats

Summary： This study investigated the effects of benazepril administered in the morning or evening on the diurnal variation of renin-angiotensin-aldosterone system （RAAS） and clock genes in the kidney. The male Wistar rat models of 5/6 subtotal nephrectomy （STNx） were established. Animals were ran- domly divided into 4 groups： sham STNx group （control）, STNx group, morning benazepril group （MB） and evening benazepril group （EB）. Benazepril was intragastfically administered at a dose of 10 mg/kg/day at 07：00 and 19：00 in the MB group and EB group respectively for 12 weeks. All the animals were synchronized to the light：dark cycle of 12：12 for 12 weeks. Systolic blood pressure （SBP）, 24-h urinary protein excretion and renal function were measured at 11 weeks. Blood samples and kidneys were collected every 4 h throughout a day to detect the expression pattern of renin activity （RA）, angio- tensin Ⅱ （Ang Ⅱ ） and aldosterone （Aid） by radioimmunoassay （RIA） and the mRNA expression profile of clock genes （bmall, dbp and per2） by real-time PCR at 12 weeks. Our results showed that no signifi- cant differences were noted in the SBP, 24-h urine protein excretion and renal function between the MB and EB groups. There were no significant differences in average Aid and RA content of a day between the MB group and EB group. The expression peak of bmall mRNA was phase-delayed by 4 to 8 h, and the diurnal variation of per2 and dbp mRNA diminished in the MB and EB groups compared with the control and STNx groups. It was concluded when the similar SBP reduction, RAAS inhibition and clock gene profile were achieved with optimal dose of benazepril, morning versus evening dosing of benazepril has the same renoprotection effects.

Effect of Xiaoaiping on the expression of circadian clock genes in human hepatoma HepG2 cells

Objective： Investigation of the effect of Xiaoaiping on the expression of circadian clock genes in human hepatoma HepG2 cells. Methods： Selecting the HepG2 cells in the logarithmic growth phase and assigning them to Xiaoaiping injection （XAP） group and control group. The two groups were treated with 75 mg/mL XAP or the same dose of normal saline. After 72 h of treatment, real-time PCR was used to detect the expression of circadian clock genes in HepG2 cells and Western Blot technology was used to detect the expression of related proteins. Results： The mRNA expression levels of PER1, NPAS2, NR1D1, and DEC1 in the XAP group was significantly higher than that in the control group （P〈 0.05）, while the mRNA expression levels of PER3, BMAL1, DEC2, and RORA were significantly lower in the XAP group than in the control group （P 〈 0.05）, and there was no significant difference between the mRNA expression levels of PER2, CRY1, CRY2, and TIM. Of course, the proteins＇ expression levels of the genes we had detected such as PERle3, CRYI-2, CLOCK, BMAL1 by Western Blot were consistent with the real-time PCR results above. Conclusion： XAP affects the expression of circadian clock genes in HepG2 cells.

Capsaicin alleviates the hepatic clock gene disruption and gut microbiota dysbiosis in circadian rhythm disorder mouse model

As the body’s internal clock,the circadian rhythm regulates the energy expenditure,appetite,and sleep.There exists a close relationship between the host circadian rhythm and gut microbiota.In this work,a circadian disorder mouse model induced by constant darkness(CD)was constructed to investigate the regulating effects of capsaicin(CAP)on disturbances of metabolism homeostasis and gut microbiota in the respect of circadian rhythm-related mechanisms.Our results indicated that CAP reduced weight gain induced by circadian rhythm disorder in mice by inhibiting fat accumulation in liver and adipose tissue.The rhythmic expressions of circadian clock genes and lipid-metabolism related genes in liver were also recovered by CAP.Microbial study using 16S rRNA sequencing revealed that CAP modulated the gut microbiota richness,diversity and composition,and restored diurnal oscillations of gut microbes at the phylum and family level.These results indicated that CAP could alleviate CD-induced hepatic clock gene disruption and gut microbiota dysbiosis in mice,providing theoretical basis for CAP to be used as a muti-functional ingredient with great healthpromoting effects.

Co-regulation of circadian clock genes and microRNAs in bone metabolism

Mammalian bone is constantly metabolized from the embryonic stage,and the maintenance of bone health depends on the dynamic balance between bone resorption and bone formation,mediated by osteoclasts and osteoblasts.It is widely recognized that circadian clock genes can regulate bone metabolism.In recent years,the regulation of bone metabolism by non-coding RNAs has become a hotspot of research.MicroRNAs can participate in bone catabolism and anabolism by targeting key factors related to bone metabolism,including circadian clock genes.However,research in this field has been conducted only in recent years and the mechanisms involved are not yet well established.Recent studies have focused on how to target circadian clock genes to treat some diseases,such as autoimmune diseases,but few have focused on the co-regulation of circadian clock genes and microRNAs in bone metabolic diseases.Therefore,in this paper we review the progress of research on the co-regulation of bone metabolism by circadian clock genes and microRNAs,aiming to provide new ideas for the prevention and treatment of bone metabolic diseases such as osteoporosis.

Circadian rhythm disruption and endocrine-related tumors

This review delved into the intricate relationship between circadian clocks and physiological processes,emphasizing their critical role in maintaining homeo-stasis.Orchestrated by interlocked clock genes,the circadian timekeeping system regulates fundamental processes like the sleep-wake cycle,energy metabolism,immune function,and cell proliferation.The central oscillator in the hypothalamic suprachiasmatic nucleus synchronizes with light-dark cycles,while peripheral tissue clocks are influenced by cues such as feeding times.Circadian disruption,linked to modern lifestyle factors like night shift work,correlates with adverse health outcomes,including metabolic syndrome,cardiovascular diseases,infec-tions,and cancer.We explored the molecular mechanisms of circadian clock genes and their impact on metabolic disorders and cancer pathogenesis.Specific associ-ations between circadian disruption and endocrine tumors,spanning breast,ovarian,testicular,prostate,thyroid,pituitary,and adrenal gland cancers,are highlighted.Shift work is associated with increased breast cancer risk,with PER genes influencing tumor progression and drug resistance.CLOCK gene expression correlates with cisplatin resistance in ovarian cancer,while factors like aging and intermittent fasting affect prostate cancer.Our review underscored the intricate interplay between circadian rhythms and cancer,involving the regulation of the cell cycle,DNA repair,metabolism,immune function,and the tumor microenvir-onment.We advocated for integrating biological timing into clinical consider-ations for personalized healthcare,proposing that understanding these connec-tions could lead to novel therapeutic approaches.Evidence supports circadian rhythm-focused therapies,particularly chronotherapy,for treating endocrine tumors.Our review called for further research to uncover detailed connections between circadian clocks and cancer,providing essential insights for targeted treatments.We emphasized the importance of public health interventions to mitigate lifestyle-related circadian disruptions and underscored the critical role of circadian rhythms in disease mechanisms and therapeutic interventions.

The Clock gene clone and its circadian rhythms in Pelteobagrus vachelli

The Clock gene,a key molecule in circadian systems,is widely distributed in the animal kingdom. We isolated a 936-bp partial c DNA sequence of the C lock gene( Pva- clock) from the darkbarbel catfish P elteobagrus vachelli that exhibited high identity with C lock genes of other species of fish and animals(65%–88%). The putative domains included a basic helix-loop-helix(b HLH) domain and two period-ARNT-single-minded(PAS) domains,which were also similar to those in other species of fish and animals. P va- Clock was primarily expressed in the brain,and was detected in all of the peripheral tissues sampled. Additionally,the pattern of P va- Clock expression over a 24-h period exhibited a circadian rhythm in the brain,liver and intestine,with the acrophase at zeitgeber time 21:35,23:00,and 23:23,respectively. Our results provide insight into the function of the molecular C lock of P. vachelli.

Hepatitis B and circadian rhythm of the liver

The circadian rhythm in humans is determined by the central clock located in the hypothalamus’s suprachiasmatic nucleus,and it synchronizes the peripheral clocks in other tissues.Circadian clock genes and clock-controlled genes exist in almost all cell types.They have an essential role in many physiological processes,including lipid metabolism in the liver,regulation of the immune system,and the severity of infections.In addition,circadian rhythm genes can stimulate the immune response of host cells to virus infection.Hepatitis B virus(HBV)infection is the leading cause of liver disease and liver cancer globally.HBV infection depends on the host cell,and hepatocyte circadian rhythm genes are associated with HBV replication,survival,and spread.The core circadian rhythm proteins,REV-ERB and brain and muscle ARNTL-like protein 1,have a crucial role in HBV replication in hepatocytes.In addition to influencing the virus’s life cycle,the circadian rhythm also affects the pharmacokinetics and efficacy of antiviral vaccines.Therefore,it is vital to apply antiviral therapy at the appropriate time of day to reduce toxicity and improve the effectiveness of antiviral treatment.For these reasons,understanding the role of the circadian rhythm in the regulation of HBV infection and host responses to the virus provides us with a new perspective of the interplay of the circadian rhythm and anti-HBV therapy.Therefore,this review emphasizes the importance of the circadian rhythm in HBV infection and the optimization of antiviral treatment based on the circadian rhythm-dependent immune response.

The clock gene,period,influences migratory flight and reproduction of the oriental armyworm,Mythimna separata(Walker)

The oriental armyworm,Mythimna separata,is a major long-distance migratory insect pest of grain crops in China and other Asian countries.Migratory flights and reproductive behavior usually occur at night,regulated by a circadian rhythm.However,knowledge about the linkages between adult flight,reproduction,and clock genes is still incomplete.To fill this important gap in our knowledge,a clock gene(designated Msper)was identified and phylogenetic analysis indicated that the encoded protein(MsPER)was highly similar to PER proteins from other insect species.Quantitative RT-PCR assays demonstrated that significantly different spatiotemporal and circadian rhythmic accumulations of mRNA encoding MsPER occurred during development under steady 14 h:10 h light:dark conditions.The highest mRNA accumulation occurred in adult antennae and the lowest in larvae.Msper was expressed rhythmically in adult antennae,relatively less in photophase and more entering scotophase.Injecting small interference RNA(siRNA)into adult heads effectively knocked down Msper mRNA levels within 72 h.Most siRNA-injected adults reduced their evening flight activity significantly and did not exhibit a normal evening peak of flight activity.They also failed to mate and lay eggs within 72 h.Adult mating behavior was restored to control levels by 72 h post injection.We infer that Msper is a prominent clock gene that acts in regulating adult migratory flight and mating behaviors of M.separata.Because of its influence on migration and mating,Msper may be a valuable gene to target for effective management of this migratory insect.

Effects of biological clock gene BMAL1 and hypoxia-inducible factor HIF-1αon proliferation,migration and radiotherapy sensitivity of nasopharyngeal carcinoma cells HONE1

Objective To understand the effects of clock gene BMAL1 and HIF-1α(Hypoxia inducible factor-1α)on proliferation,migration and sensitivity to radiotherapy of nasopharyngeal carcinoma cells HONE1.At the same time,whether the biological clock gene BMAL1 can affect the expression of HIF-1αprotein was investigated.It will lay the foundation for further study on the correlation between clock gene BMAL1 and HIF pathway.Methods BMAL1 gene overexpression and interference lentivirus and HIF-1αgene interference lentivirus were constructed respectively,and were transfected into nasopharyngeal carcinoma cells HONE1.Western blot was used to verify the establishment of overexpressed and knockdown BMAL1 cell lines and HIF-1αgene knockdown cell line,and to investigate the expression of HIF-1αprotein in overexpressed and knockdown BMAL1 cell lines.CCK-8 cell proliferation test and scratch test were used to analyze the proliferation and migration ability of cells.Cell apoptosis after radiotherapy was analyzed by flow cytometry.The effects of BMAL1 and HIF-1αon the sensitivity of HONE1 radiotherapy in nasopharyngeal carcinoma cells after X-ray irradiation at different doses(0Gy,2Gy,4Gy,6Gy)were detected by clone formation assay.Results The overexpression of BMAL1 gene and lentivirus interference were constructed to effectively up regulate and down regulate the expression of BMAL1 protein in nasopharyngeal carcinoma cells HONE1.Meanwhile,HIF-1αgene interference lentivirus was constructed to effectively down-regulate the expression of HIF-1αprotein in nasopharyngeal carcinoma cell line HONE1,and successfully screen out stable nasopharyngeal carcinoma cell lines.Western blot results showed that overexpression of BMAL1 gene could inhibit the expression of HIF-1αprotein in HONE1 of nasopharyngeal carcinoma cells,while knockdown of BMAL1 gene promoted the expression of HIF-1αprotein in HONE1 of nasopharyngeal carcinoma cells(P<0.05).CCK-8 cell proliferation and scratch test showed that overexpression of BMAL1 gene or knockdown of HIF-1αgene could inhibit the proliferation and migration of HONE1 cells(P<0.05).Flow cytometry results showed that after 8Gy irradiation for 72 h,the apoptosis rate of BMALl gene overexpression group was higher than that of the overexpression control group,similarly,the apoptosis rate of HIF-1αgene knockdown group was higher than that of the knockdown control group(P<0.05).After X-ray irradiation at different doses(0Gy,2Gy,4Gy,6Gy),clon-formation experiment showed that the clon-formation rate and cell survival fraction of BMALl overexpression group or HIF-1αknockdown group were lower than those of negative control group(P<0.05).Sigmaplot analysis showed that the D0,Dq and SF2 of the BMAL1 overexpression group or HIF-1αknockdown group were lower than those of the negative control group,and the radiosensitization ratios were 1.381 and 1.063,respectively.Conclusion Overexpression of BMAL1 gene can inhibit the proliferation and migration of nasopharyngeal carcinoma cell line HONE1,increase apoptosis after radiotherapy and improve radiosensitivity.Knock down HIF-1αGene can inhibit the proliferation and migration of nasopharyngeal carcinoma cell line HONE1,increase apoptosis after radiotherapy and improve radiosensitivity.In nasopharyngeal carcinoma cells HONE1,overexpression of BMAL1 gene can inhibit the expression of HIF-1αprotein while knockdown of BMAL1 gene can promote the expression of HIF-1αprotein.

Hibernation with rhythmicity:the circadian clock and hormonal adaptations of the hibernating Asiatic toads(Bufo gargarizans)

Hibernation is one of the fundamental strategies in response to cold environmental temperatures.During hibernation,the endocrine and circadian systems ensure minimal expenditure of energy for survival.The circadian rhythms of key hormones,melatonin(MT),corticosterone(CORT),triiodothyronine(T3),and thyroxine(T4),and the underlying molecular regulatory mechanisms of hibernation have been well determined in mammals but not in ectotherms.Here,a terrestrial hibernating species,Asiatic toad(Bufo gargarizans),was employed to investigate the plasma CORT,MT,T3,and T4;and the retina,brain,and liver mRNA expression of the core clock genes,including circadian locomotor output cycles kaput(Clock),brain and muscle ARNT-like 1(Bmal1),cryptochrome(Cry)1 and 2,and period(Per)1 and 2,at 7-time points over a 24-h period under acute cold(1 day at 4℃),and hibernation(45 days at 4℃).Our results showed that the circadian rhythms of the core clock genes were rather unaffected by acute cold exposure in the retina,unlike the brain and liver.In contrast,during hibernation,the liver clock genes displayed significant circadian oscillations,while those in the retina and brain stopped ticking.Furthermore,plasma CORT expressed circadian oscillations in both groups,and T3 in acute cold exposure group,whereas T4 and MT did not.Our results reveal that the plasma CORT and the liver sustain rhythmicity when the brain was not,indicating that the liver clock along with the adrenal clock synergistically maintains the metabolic requirements to ensure basic survival in hibernating Asiatic toads.

Role of circadian gene Clock during differentiation of mouse pluripotent stem cells

Biological rhythms controlled by the circadian clock are absent in embryonic stem cells （ESCs）. However, they start to develop during the differentiation of pluripotent ESCs to downstream cells. Conversely, biological rhythms in adult somatic cells disappear when they are reprogrammed into induced pluripotent stem cells （iPSCs）. These studies indicated that the development of biological rhythms in ESCs might be closely associated with the maintenance and differentiation of ESCs. The core circadian gene Clock is essential for regulation of biological rhythms. Its role in the development of biological rhythms of ESCs is totally unknown. Here, we used CRISPR/CAS9-mediated genetic editing techniques, to completely knock out the Clock expression in mouse ESCs. By AP, teratoma formation, quantitative real-time PCR and Immunofluorescent staining, we did not find any dif- ference between Clock knockout mESCs and wild type mESCs in morphology and pluripotent capability under the pluripotent state. In brief, these data indicated Clock did not influence the maintaining of pluripotent state. However, they exhibited decreased proliferation and increased apoptosis. Furthermore, the biological rhythms failed to develop in Clock knockout mESCs after spontaneous differentiation, which indicated that there was no compensational factor in most peripheral tissues as described in mice models before （DeBruyne et ah, 2007b）. After spontaneous differentiation, loss of CLOCK protein due to Clock gene silencing induced spontaneous differentiation of mESCs, indicating an exit from the pluripotent state, or its differentiating ability. Our findings indicate that the core circadian gene Clock may be essential during normal mESCs differentiation by regulating mESCs proliferation, apoptosis and activity.

Understanding light pollution: Recent advances on its health threats and regulations

The prevalence of artificial lights not only improves the lighting conditions for modern society,but also poses kinds of health threats to human health.Although there are regulations and standards concerning light pollution,few of them are based on the potential contribution of improper lighting to diseases.Therefore,a better understanding of the health threats induced by light pollution may promote risk assessment and better regulation of artificial lights,thereby a healthy lighting environment.This review is based on a careful collection of the latest papers from 2018 to 2022 about the health threats of light pollution,both epidemiologically and experimentally.In addition to summing up the novel associations of light pollution with obesity,mental disorders,cancer,etc.,we highlight the toxicological mechanism of light pollution via circadian disruption,since light pollution directly interferes with the natural light-dark cycles,and damages the circadian photoentrainment of organisms.And by reviewing the alternations of clock genes and disturbance of melatonin homeostasis induced by artificial lights,we aim to excavate the profound impacts of light pollution based on accumulating studies,thus providing perspectives for future research and guiding relevant regulations and standards.

Exposure to endocrine disrupting chemicals perturbs lipid metabolism and circadian rhythms

A growing body of evidence indicates that exposure to environmental chemicals can contribute to the etiology of obesity by inappropriately stimulating adipogenesis as well as perturbing lipid metabolism and energy balance. One potential mechanism by which chemical exposure can influence lipid metabolism is through disturbance of circadian rhythms, endogenously-driven cycles of roughly 24 hr in length that coordinate biochemical, physiological, and behavioral processes in all organisms. Here we show for the first time that exposure to endocrine disrupting compounds（EDCs）, including the pesticide tributyltin, two commercial flame retardants, and a UV-filter chemical found in sunscreens,can perturb both circadian clocks and lipid metabolism in vertebrates. Exposure of developing zebrafish to EDCs affects core clock activity and leads to a remarkable increase in lipid accumulation that is reminiscent of the effects observed for longdaysin, a known disruptor of circadian rhythms. Our data reveal a novel obesogenic mechanism of action for environmental chemicals, an observation which warrants further research. Because circadian clocks regulate a wide variety of physiological processes, identification of environmental chemicals capable of perturbing these systems may provide important insights into the development of environmentally-induced metabolic disease.